RNA molecules are secreted in extracellular spaces (exRNAs) and act as endocrine signals altering the phenotypes of cancer cells. In this application we will focus on the class of small non-codingRNAs named microRNAs. We propose an innovative therapeutic concept: the depletion of oncogenic microRNAs from malignant cells by enhancing their secretion as exRNAs or blocking suppressor microRNAs' delivery in extracellular space by blocking secretion mechanisms. Our main goal is to develop new strategies using combined miRNA and siRNA to maximize therapeutic benefit by affecting the production of exRNAs. Our proposal centers on the identification and targeting of specific tumor-derived exRNA and exosomes leading to novel therapies and improved therapeutic outcomes. In UH2 phase of the grant, using ovarian cancer (OC) cell lines and cells derived from OC patient tumors, we will identify novel exRNA-based cancer therapeutic lead candidates by siRNA library genome-wide screening based on secretion measurements, by small RNA deep sequencing in paired samples from tumors, plasma and ascites from the same patient, and by functional studies for the lead exRNA candidates. In the UH3 phase, we will achieve pre-clinical optimization by testing the exRNA lead(s) identified in the UH2 for antitumor efficacy in well-established OC murine orthotopic models and their safety using well-established preclinical protocols. The aims in exploratory UH2 phase are designed to identify and validate the function of novel exRNA therapeutic candidates for pre- clinical studies. In UH3 phase, we will formulate the methods for the validation of the assays to determine the activity, pharmacology in tissue and plasma to establish dosage schedules in animal models for the design of rational anti-tumor approaches. Using our nanoliposomal delivery technology, we will also conduct target modulation and efficacy studies to generate clear evidence regarding the safety of exRNA-based therapeutic candidates in the proposed dose range. Our approach, by using a new category of regulatory exRNAs- miRNAs, could substantially enhance therapeutic efficacy for cancer treatment. We will also demonstrate, for the first time, the use of nanoliposome-siRNA-exRNA targeting to generate a synergistic boosting effect for treatment of cancers. While we are focusing on OC due to the high mortality associated with this malignancy, our findings have applications for any type of cancer. This proposal brings synergistic capabilities of scientists working for over a decade in the fields of miRNA (Dr. Calin), siRNA (Dr. Sood), nanoparticle delivery (Dr. Lopez-Berestein), and exosome biology (Dr. O'Halloran). The team has the expertise and synergistic drive to achieve novel exRNA therapeutic development by performing highly innovative research, and has developed RNA-based therapies in the past.